Engineering AAV for safe and efficient gene delivery to the human retina

摘要 这是R 01 EY 024280的竞争性更新，“开发用于光受体靶向的高效AAV载体 通过维塔尔”。请注意，由于在最初供资期间取得的预付款， 为了了解该领域的挑战，我们的标题已改为“安全高效的工程AAV” 将基因传递到人类视网膜”，以更广泛地涵盖我们的目标。FDA批准Adeno相关 基于病毒（AAV）的RPE 65-Leber先天性黑蒙（LCA 2）基因治疗巩固了基因治疗的地位 在目前的医疗实践中。然而，在一些患者的中心凹下注射载体导致中央视网膜病变。 变薄和视力丧失。在无脉络膜症中也观察到类似的视网膜厚度减少 临床试验在更严重的情况下，如X连锁视网膜劈裂症（XLRS），人们担心视网膜下 注射（SRI）将进一步损害患者视网膜。由于大多数遗传性视网膜疾病（IRD）是由 感光细胞（PR）和视网膜色素上皮（RPE）特异性基因突变，基因发育 更安全和有效地靶向这些细胞的治疗仍然是一个重要的、未满足的需求。靶向中心凹 视锥细胞尤其重要，因为它们负责敏锐的日光视觉。在最初的筹资期间， 我们开发了在灵长类动物中玻璃体内注射（IVI）后能够有效视网膜转导的AAV衣壳。 内界膜（ILM）是AAV通过该途径转导的主要屏障。然而，结果 来自利用IVI AAV的临床试验，其显示出剂量限制性炎症和AAV衣壳的中和 预先存在的抗体（NAb）暗示宿主免疫系统是临床免疫的更直接的“屏障”。 翻译.眼睛的“免疫特权”可能导致了对免疫系统作用的低估 在塑造眼内递送的AAV的结果方面。天然存在的抗衣壳抗体能够 通过玻璃体转导视网膜（即AAV 2）在高达70%的人中普遍存在。因此，很大一部分 的患者不符合新兴疗法的入选标准。在这里，我们提出的实验，基于强 初步数据，以克服这些障碍。大部分工作将在灵长类动物（猕猴）中进行， 这些屏障只能在具有眼部特征和免疫系统的动物的完整眼睛中重现 类似于人类。在目标1中，我们将通过以下方式增强玻璃体内递送的AAV的转导和安全性： 改造衣壳和基因组以避免免疫识别。在目标2中，我们将增强视网膜转导 通过在ILM下递送AAV来实现内部和外部视网膜的有效和特异性转导。在目标3中， 将增强通过视网膜下递送的AAV的转导，所述AAV横向扩散超过注射部位。向量 本提案中研究的方法将对计划的临床试验产生直接影响， 遗传性视网膜疾病以及非孤儿适应症如AMD。开发这些工具， 学术界（而不是工业界）将确保与更广泛的科学界共享资源的可用性。 社区

ABSTRACT This is a competitive renewal of R01EY024280, “Developing Efficient AAV Vectors for Photoreceptor Targeting via the Vitreous”. Note that, due to advances made during the initial funding period, and our improved understanding of challenges in the field, our title has been changed to “Engineering AAV for safe and efficient gene delivery to the human retina” to more broadly encompass our goals. FDA approval of an Adeno associated virus (AAV)- based gene therapy for RPE65-Leber congenital amaurosis (LCA2) solidified gene therapy’s place in current medical practice. However, injection of vector under the fovea of some patients led to central retinal thinning and loss of visual acuity. Similar decreases in retinal thickness were also observed in Choroideremia clinical trials. In more severe conditions, like X-linked Retinoschisis (XLRS), there is concern that subretinal injection (SRI) will further damage patient retinas. Since most inherited retinal diseases (IRDs) are caused by mutations in photoreceptor (PR)- and retinal pigment epithelial (RPE)- specific genes, development of gene therapies that more safely and efficiently target these cells remains a significant, unmet need. Targeting foveal cones is especially important, as they are responsible for acute, daylight vision. During the initial funding period, we developed AAV capsids capable of efficient retinal transduction following intravitreal injection (IVI) in primate. The inner limiting membrane (ILM) is the major barrier to AAV transduction via this this route. However, results from clinical trials utilizing IVI AAVs that show dose-limiting inflammation, and neutralization of the AAV capsid by pre-existing antibodies (NAbs) implicate the host immune system as a more immediate ‘barrier’ to clinical translation. The eye’s ‘immune-privilege’ has perhaps led to an under appreciation of the immune system’s role in shaping the outcome of intra-ocularly delivered AAVs. Naturally occurring antibodies to capsids capable of transducing retina via the vitreous (i.e. AAV2) are prevalent in up to 70% of humans. As such, a large percentage of patients will not meet inclusion criteria for emerging therapies. Here we propose experiments, based on strong preliminary data, to overcome these barriers. The majority of work will be performed in primates (macaque) as these barriers can only be recapitulated in intact eyes of animals with ocular characteristics and immune systems similar to humans. In Aim 1, we will enhance transduction and safety of intravitreally delivered AAVs by engineering the capsid and genome to avoid immune recognition. In Aim 2, we will enhance retinal transduction by subILM delivery of AAVs to enable efficient and specific transduction of inner and outer retina. In Aim 3, we will enhance transduction by subretinally delivered AAVs that spread laterally beyond the injection site. Vectors and methods investigated in this proposal will have an immediate impact on planned clinical trials to address inherited retinal diseases as well as non-orphan indications such as AMD. Development of these tools by academia (rather than industry) will ensure the availability of shared resources with the broader scientific community.